The ability of platelets to rapidly stick to the damaged wall of arterial blood vessels is critical for preventing blood loss (hemorrhage). Inappropriate deposition of these hemostatic cells in arterial blood vessels due to pathological disease processes such as artherosclerosis can result in lack of blood flow to vital organs such as the heart and brain. Thus a delicate balance exists between providing adequate hemostasis without causing blockage of blood vessels by excessive platelet deposition (a.k.a. thrombus formation).
von Willebrand Factor (VWF) is a multidomain, plasma glycoprotein of complex multimeric structure which is synthesized by vascular endothelial cells and megakaryocytes (1-3) (FIG. 1A). Its presence in the blood is vital to maintaining the integrity of the vasculature. To accomplish this task, VWF forms a “bridge” between the injured vessel wall and platelets by virtue of its ability to interact with extracellular matrix components, such as collagen, and receptors expressed on platelets, such as glycoprotein Ib alpha (4-9). It also binds to and confers stability to factor VIII (10). The importance of this glycoprotein in hemostasis is underscored by the occurrence of clinical bleeding when the plasma VWF levels fall below 50 IU/dL (type I von Willebrand's disease, VWD), or when functional defects in the protein occur (type 2 VWD) (11,12).
Upon surface immobilization of VWF at sites of vascular injury, it is the role of the A1 domain of VWF (residues 1260-1480) to initiate the process of platelet deposition at sites of vascular injury and under conditions of high rates of shear flow (>1,000 s−1; Ruggeri, Z. M. et al., Blood. 108, 1903-1910 (2006)). The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M VWD, which results from the incorporation of loss-of-function mutations within this domain that perturb interactions with GPIb alpha (Sadler, J. E. et al. (2006) J. Thromb. Haemost. 4, 2103-2114; Rabinowitz, I. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9846-9849; Cruz et al., (2000) J. Biol. Chem. 275, 19098-19105). In addition, recombinant VWF multimers lacking the A1 domain cannot support platelet adhesion at high rates of flow despite retaining the ability to interact with collagen (Sixma et al., (1991) Eur J Biochem. 196:369-75).
The structure of the A1 domain includes the α/β fold with a central β-sheet flanked by α-helices on each side as well as one intra-disulfide bond (Cys1272-Cys 1458), but no MIDAS motif (Emsley et al., (1998) J Biol Chem. 273:10396-401). Its overall shape is cuboid, with the top and bottom faces forming the major and minor binding sites, respectively, that interact with the concave surface of GPIbα. The most extensive contact site buries ˜1700 Å2 of surface area, interacting with LRR five to eight and the C-terminal flank of the GPIbα (Huizinga, E. G. et al. (2002) Science 297, 1176-1179). For this to occur, the β-switch region of this platelet receptor undergoes a conformation change so that it aligns itself with the central beta sheet of the A1 domain. The smaller site (˜900 Å2) accommodates the binding of the β-finger and the first LRR of GPIbα, an event that appears to require the displacement of the amino-terminal extension of the A1 domain. Based on these findings as well as the preferential localization of mutations in humans within this region, which enhance GPIbα binding, it is speculated that the amino-terminal extension regulates the adhesive properties of this domain. This is also supported by the fact that recombinant A1 proteins lacking this extension have a higher affinity for this platelet receptor (Sugimoto et al., (1993) J Biol Chem. 268:12185-92). Despite these observations, the physiological relevance of such structural changes in this receptor-ligand pair remains to be determined as well as the contribution of other domains to this process.
In addition to its role in hemostasis, VWF also contributes to pathological thrombus formation on the arterial side of the circulation. This may be the consequence of injury to the blood vessel wall from inflammatory disease states and/or medical/surgical interventions. Pathological thrombus formation is the leading cause of death in the Western world. Thus, pharmaceutical companies have committed considerable resources towards the research and design of drugs to prevent or treat thrombosis. However, there remains an urgent need to develop new and improved therapies such as those aimed at reducing platelet and/or VWF interactions with the injured arterial wall. One major hurdle hindering drug development in this field is the lack of an appropriate small animal model of thrombosis to test promising therapies. For instance, differences in the structure or isoform of protein receptors or ligands on mouse vs. human platelets that are critical for the activation and/or binding of these cells to the injured vessel wall preclude testing of drugs developed against human platelets in a mouse model of thrombosis. Moreover, this issue cannot be overcome by simply transfusing mice with human platelets as we have observed that mouse VWF does not support significant interactions with human cells (see below). Thus, the development of a “humanized” mouse model of hemostasis and thrombosis would potentially expedite drug discovery and testing.
That said, we have discovered that only one amino acid difference between mouse and human VWF-A1 domains accounts for most of the inability of the former to interact with human platelets and vice versa. With this knowledge in hand, we have genetically altered a mouse to express VWF that contains this amino acid found in human VWF-A1, imparting on it the ability to support adhesion of human platelets to a level observed for its human counterpart. As a result, not only are we uniquely poised to better understand the molecular mechanisms governing human platelet binding at sites of vascular injury in vivo, but now have the capability to perform pre-clinical testing of anti-thrombotic agents and targeted molecular imaging agents directed against human platelet cells in a living animal. The material contained within this document describes the features of this unique biological platform for drug testing the testing of drugs and targeted molecular imaging agents.